The determination of tissue‐specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing

Abstract

The goal of this study is to explore the application of epigenetic markers in the identification of biofluids that are commonly found at the crime scene. A series of genetic loci were examined in order to define epigenetic markers that display differential methylation patterns between blood, saliva, semen, and epithelial tissue. Among the different loci tested, we have identified a panel of markers, C20orf117, ZC3H12D, BCAS4, and FGF7, that can be used in the determination of these four tissue types. Since methylation modifications occur at cytosine bases that are immediately followed by guanine bases (CpG sites), methylation levels were measured at CpG sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated CpG-associated cytosine bases to thymine bases. The bisulfite modified DNA was then amplified via nested PCR using a primer set of which one primer was biotin labeled. Biotinylated PCR products were in turn analyzed and the methylation level at each CpG site was quantitated by pyrosequencing. The percent methylation values at each CpG site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC3H12D and FGF7 loci differentiated sperm from blood, saliva, and epithelial cells. The C20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS4. The results of this study demonstrate the applicability of epigenetic markers as a novel tool for the determination of biofluids using bisulfite modification and pyrosequencing.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood

“…To date, many CpG markers that show differential DNA methylation patterns in different types of body fluids have been identified through genome-wide DNA methylation profiling and gene-specific analysis [10][11][12][13][14][15]. Body fluid-specific hypo-or hypermethylation status, which does not give "on or off" signal, has attracted criticism when used for analysis of mixed samples, but 3 recent studies reported a set of CpG markers that show a methylation signal only in the target body fluids using Illumina's BeadChip array [13][14][15].…”

Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system

“…One method is to treat the DNA with sodium bisulfite as this changes un-methylated cytosine into uracil while methylated cytosine remains unaffected [14]. The methylation status of any PCR product can be determined by the presence of either thymine, representing a lack of methylation, or the presence of a cytosine, indicating methylation; this can be detected by pyrosequencing [15][16][17][18] or single-baseextension (SBE) [19][20][21][22]. Bisulfite conversion can lead to the undesired side-effect of DNA degradation [23], which is not ideal if the sample is low template in nature or of poor quality.…”

LINE-1 DNA methylation: A potential forensic marker for discriminating monozygotic twins

“…The assay was tested on 50 DNA samples from blood, saliva, semen, and skin epidermis, and the source tissue was successfully identified in all cases. Since then, several studies have investigated novel DNA methylation markers for identification of body fluids or tissue for use in forensic and developed new method for semen identification using DNA methylation analysis [21][22][23][24].…”